Enzyme catalyzed reaction Pseudomonas cepacia lipase

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 1 reaction 2). These enzymes also catalyze transesterifications and the asymmetrization of meso -substrates (Section 13.2.3.1.1). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities. As more and more research has been conducted with these enzymes, a less empirical approach is being taken due to the different substrate profiles amassed for various enzymes. These profiles have been used to construct active site models for such enzymes as pig liver esterase (PLE) (EC 3.1.1.1) and the microbial lipases (EC 3.1.1.3) Pseudomonas cepacia lipase (PCL), formerly P.fluorescens lipase, Candida rugosa lipase (CRL), formerly C. cylindracea lipase, lipase SAM-2 from Pseudomonas sp., and Rhizopus oryzae lipase (ROL) [108-116]. In addition, x-ray crystal structure information on PCL and CRL has been most helpful in predicting substrate activities and isomer preferences [117-119]. 

Of course, the influence of organic solvents on enzyme enantioselectivity is not limited to proteases but it is a general phenomenon. Quite soon, different research groups described the results obtained with lipases [28]. For instance, the resolution of the mucolytic drug ( )-trans-sobrerol (11) was achieved by transesteriflcation with vinyl acetate catalyzed by the lipase from Pseudomonas cepacia adsorbed on celite in various solvents. As depicted in Scheme 1.3 and Table 1.5, it was found that t-amyl alcohol was the solvent of choice in this medium, the selectivity was so high ( >500) that the reaction stopped spontaneously at 50% conversion giving both +)4rans-sobrerol and (—)-trans-sobrerol monoacetate in 100% optical purity [29]. 

Lipase-catalyzed transesterification of (3-nitroalcohol substrates had not previously been reported and required careful optimization of the reaction conditions. A series of enzymes were screened, followed by acyl donors. From these results, the lipase Pseudomonas cepacia (PS-C I) (for more... 

In contrast to the poor reaction rates reported elsewhere [109], crude powder of lipase from Pseudomonas cepacia (PSL) acylates secondary phenylethyl alcohol at reasonable reaction rates [120] (Table 4). Based on active enzyme protein, the highest reaction rates were observed for carrier-fixed enzymes. This is also true for lipase from Candida antarctica (CAL-B). PSL crystals catalyzed with comparable activity when they had been pretreated with surfactants [109]. 

Lipases are a family of enzymes that, in addition to their hydrolytic activity on triglycerides, also catalyze (trans)esterification reactions. They recognize a broad range of unnatural substrates in either aqueous or nonaqueous phase, have a high commercial availability, do not require expensive cofactors, and are easily recoverable. These factors make lipases especially interesting and they have been used extensively in, for example, asymmetric synthesis. The lipase from Pseudomonas cepacia was also targeted in a dynamic combinatorial resolution-type protocol [36]. Based on the efficient nitroaldol (Henry) reaction, DCLs of aldehydes, nitroal-kanes, and P-nitroalcohols could be easily generated (Scheme 5.9). 

In an alternate process for the preparation of C-13 taxol side chain, the stereoselective enzymatic hydrolysis of racemic ci5-3-(acetyloxy)-4-phenyl-2-azetidinone 42 to the corresponding (S)-(—)-alcohol 43 has been demonstrated [53,54]. Lipase PS-30 from Pseudomonas cepacia (Amano International Enzyme Co.) and BMS lipase (extracellular lipase derived from the fermentation of Pseudomonas sp. SC 13856) catalyzed hydrolysis of the undesired enantiomer of racemic 42, producing S-(—)-alcohol 43 and the desired i -(+)-acetate 44 (Fig. 12). Reaction yields of more than 48% (theoretical maximum yield is 50%) and e.e. of more than 99.5% were obtained for the desired R-(+)-acetate. For a very efficient enzyme source (BMS lipase), a lipase fermentation using Pseudomonas sp. SG 13865 was developed. In a fed-batch process using soybean oil, the fermentation resulted in 1500 U/ml of extracellular lipase activity. Crude BMS lipase (1.7 kg containing 140,000 U/g) was recovered from the filtrate by ethanol precipitation. BMS lipase and lipase PS-30 were immobilized on Accurel polypropylene (PP). These immobilized lipases were reused (10 cycles) without loss of enzyme activity, productivity, or the e.e. of the product in the resolution process. The enzymatic process for the resolution of racemic acetate 42 was scaled up to 150 L at 10 g/L substrate concentration using inunobilized BMS lipase and lipase PS-30, respectively. From each reaction batch, 3-(R)-acetate 44 was isolated in 45 M% yield (theoretical maximum yield is 50%) and 99.5% e.e. 3-(R)-acetate 44 was chemically converted to 3-(R)-alcohol 45. The C-13 taxol side chain (41a or 45) produced... 

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